1-Methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity has previously been attributed to either caspase-dependent apoptosis or caspase-independent cell death. In the current study, we found that MPP(+) induces a unique, non-apoptotic nuclear morphology coupled with a caspase-independent but calpain-dependent mechanism of cell death in primary cultures of rat cerebellar granule neurons (CGNs). Using a terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay in CGNs exposed to MPP(+), we observed that these neurons are essentially devoid of caspase-dependent DNA fragments indicative of apoptosis. Moreover, proteolysis of a well recognized caspase-3 substrate, poly (ADP ribose) polymerase (PARP), was not observed in CGNs exposed to MPP(+). In contrast, calpain-dependent proteolysis of fodrin and pro-caspases-9 and -3 occurred in this model coupled with inhibition of caspase-3/-7 activities. Notably, several key members of the Bcl-2 protein family appear to be prominent calpain targets in MPP(+)-treated CGNs. Bid and Bax were proteolyzed to truncated forms thought to have greater pro-death activity at mitochondria. Moreover, the pro-survival Bcl-2 protein was degraded to a form predicted to be inactive at mitochondria. Cyclin E was also cleaved by calpain to an active low MW fragment capable of facilitating cell cycle re-entry. Finally, MPP(+)-induced neurotoxicity in CGNs was significantly attenuated by a cocktail of calpain and caspase inhibitors in combination with the antioxidant glutathione. Collectively, these results demonstrate that caspases do not play a central role in CGN toxicity induced by exposure to MPP(+), whereas calpain cleavage of key protein targets, coupled with oxidative stress, plays a critical role in MPP(+)-induced neurotoxicity. Our findings underscore the complexity of MPP(+)-induced neurotoxicity and suggest that calpain may play a fundamental role in causing neuronal death downstream of mitochondrial oxidative stress and dysfunction.